The long range goal of this project is to understand structure- function relationships for a metabolic enzyme involved in the salvage of purine bases. The enzyme that is the subject of this study is the hypoxanthine phosphoribosyltransferase (HPRT) of Trypanosoma cruzi, etiologic agent of Chagas' disease. In humans, de novo, as well as salvage pathways exist for the synthesis of purine nucleotides. However, the complete absence HPRT activity is responsible for Lesch-Nyhan syndrome, while a partial deficiency can result in gouty arthritis. In contrast, most parasites are unable to synthesize purines via de novo pathways, and must rely on enzymes in salvage pathways, including HPRTs, for the purines needed in cellular metabolism. Thus, HPRTs have been identified as potential targets for drugs in the chemotherapeutic treatment of human disease caused by several species of parasites. Recently, two high resolution crystal structures of the trypanosomal HPRT were solved in our laboratory - a 1.4 Angstrom units resolution structure of the enzyme co-crystallized with a product analog (Focia, et al. - a) and a 1.8 Angstrom units resolution structure of the enzyme captured in a closed, pre-transition state conformation with the primary substrate, phosphoribosylpyrophosphate (PRPP) and a hypoxanthine analog (Focia, et al. - b). These structures provide snapshots of an HPRT at different stages of the enzyme-catalyzed reaction and enable predictions for the roles of specific amino acids in the chemistry of the reaction. For the studies presented herein, site-specific replacement and saturation mutagenesis of the cloned gene, coupled with kinetic and structural studies of the resultant mutant enzymes, will be used to test the structure-based predictions. Recently, a novel system was developed in our laboratory that enables the selection for active recombinant HPRTs by complementation in bacteria (Canyuk et al., in press). This assay will be used to provide a rapid assessment of the functional role(s) for target amino acids by selecting from random libraries of mutant HPRTs created by saturation mutagenesis, those enzymes with sufficient activity to rescue the genetically deficient bacteria. Target amino acids in the trypanosomal enzyme chosen to illuminate details of the catalytic mechanism of HPRTs will include residues that 1) form a flexible loop demonstrated to close over the active site, 2) flank a conserved non-proline cis-peptide on the floor of the active site, and 3) interact directly with bound substrates and/or metal ions. Selected mutant forms of the enzyme will be characterized kinetically, using steady-state and physical binding methods, and where appropriate, crystal structures of the mutant enzymes will be determined. The results of this study will greatly enhance our understanding of structure-function relationships for this important metabolic enzyme. Benefits for the public include a better understanding of the molecular basis of human disease as well as the provision of information that could be used in strategies for the design of drugs to treat diseases which are a significant burden to human kind.